scholarly journals Transient increase in calcium efflux accompanies fertilization in Chlamydomonas.

1983 ◽  
Vol 97 (2) ◽  
pp. 397-404 ◽  
Author(s):  
R A Bloodgood ◽  
E N Levin
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Mating Type ◽  
Transient Increase ◽  
Calcium Efflux ◽  
Efflux Rate ◽  
Mating Structure ◽  
Flagellar Membrane ◽  
Intracellular Storage ◽  
Cell Cell

Mating in Chlamydomonas is a complex process initiated by contact of gametic flagellar surfaces, resulting in transmission of a signal from the flagella to the cell bodies. This signal triggers later events of cell wall loss, mating structure activation, and cell-cell fusion. Little is known about the nature of the signal or the role of Ca in these events. It was found that extracellular Ca is not necessary for successful mating in Chlamydomonas. However, cells will take up Ca from the medium in a linear manner for many hours and will accumulate micromolar concentrations, presumably by sequestering Ca within intracellular storage sites. If gametic cells of one mating type (preloaded with 45Ca) are mated with gametes of the opposite mating type (preloaded with unlabeled calcium), there is a rapid, transient increase in calcium efflux rate (20 times that of the control) that lasts approximately 6 min. This effect is not associated with cell-cell fusion, since the same observation is made if (+) gametes preloaded with 45-Ca are agglutinated by isolated flagella from (-) gametes preloaded with unlabeled Ca. Other experiments have shown that the increased efflux rate is not a simple consequence of cell wall release. Ca efflux in unmated gametes is greatly reduced in deflagellated cells, suggesting that much of the Ca movement is associated with the flagellar membrane. Although signaling itself may involve Ca fluxes across the flagellar membrane, it is also possible that a consequence of signaling is release of Ca from intracellular storage sites (perhaps functional equivalents of the sarcoplasmic reticulum). The observed transient increase in Ca efflux rate may reflect a transient increase in the cytoplasmic free-Ca concentration. This increase in cytoplasmic Ca may regulate the later events in mating (such as cell wall release and mating structure activation).

scholarly journals The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

1997 ◽  
Vol 137 (7) ◽  
pp. 1537-1553 ◽  
Author(s):  
Nedra F. Wilson ◽  
Mary J. Foglesong ◽  
William J. Snell
Keyword(s):  
Cell Fusion ◽  
Mating Type ◽  
Plasma Membranes ◽  
Surface Proteins ◽  
Filamentous Actin ◽  
Mating Structure ◽  
Cell Protrusion ◽  
Isolation And Characterization ◽  
In Vitro Adhesion

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ∼3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 106 fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes.

scholarly journals Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

2021 ◽  
Author(s):  
Olivia Muriel ◽  
Laetitia Michon ◽  
Wanda Kukulski ◽  
Sophie G Martin
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Sexual Reproduction ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Turgor Pressure ◽  
Wall Material ◽  
M Cells ◽  
P Cells ◽  
Cell Cell

Cell-cell fusion is central to the process of fertilization for sexual reproduction. This necessitates the remodeling of peri-cellular matrix or cell wall material and the merging of plasma membranes. In walled fission yeast S. pombe, the fusion of P and M cells during sexual reproduction relies on the fusion focus, an actin structure that concentrates glucanase-containing secretory vesicles for local cell wall digestion necessary for membrane fusion. Here, we present a correlative light and electron microscopy (CLEM) quantitative study of a large dataset of 3D tomograms of the fusion site, which revealed the ultrastructure of the fusion focus as an actin-containing, vesicle-dense structure excluding other organelles. Unexpectedly, the data revealed asymmetries between the two gametes: M-cells exhibit a taut and convex plasma membrane that progressively protrudes into P-cells, which exhibit a more slack, wavy plasma membrane. These asymmetries are relaxed upon plasma membrane fusion, with observations of ramified pores that may result from multiple initiations or inhomogeneous expansion. We show that P-cells have a higher exo- to endocytosis ratio than M-cells, and that local reduction in exocytosis abrogates membrane waviness and compromises cell fusion significantly more in P- than M-cells. Reciprocally, reduction of turgor pressure specifically in M-cells prevents their protrusions into P-cells and delays cell fusion. Thus, asymmetric membrane conformations, which result from differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.

scholarly journals ISOLATION AND GENETIC ANALYSIS OF MUTANT STRAINS OF CHLAMYDOMONAS REINHARDI DEFECTIVE IN GAMETIC DIFFERENTIATION

Genetics ◽  
1976 ◽  
Vol 82 (2) ◽  
pp. 169-186
Author(s):  
Ursula W Goodenough ◽  
Carol Hwang ◽  
Howard Martin
Keyword(s):  
Genetic Analysis ◽  
Cell Fusion ◽  
Mating Type ◽  
Normal Growth ◽  
The Other ◽  
Genetic Dissection ◽  
Structural Studies ◽  
Tetrad Analysis ◽  
Mating Structure ◽  
Mutant Strains

ABSTRACT Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-6, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt  - or mt  + gametes; these strains have been induced to form rare zygotes with mt  - gametes and the mutations are shown to be unlinked to the mt locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-1 strain agglutinate well with mt  - gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt  + locus, and fine-structural studies reveal that imp-1gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.

scholarly journals Osmotic Balance Regulates Cell Fusion during Mating in Saccharomyces cerevisiae

1997 ◽  
Vol 138 (5) ◽  
pp. 961-974 ◽  
Author(s):  
Jennifer Philips ◽  
Ira Herskowitz
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Cell Contact ◽  
Fusion Event ◽  
Glycerol Facilitator ◽  
Fusion Defect ◽  
Osmotic Balance ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Gpd1 Gene ◽  
Cell Cell

Successful zygote formation during yeast mating requires cell fusion of the two haploid mating partners. To ensure that cells do not lyse as they remodel their cell wall, the fusion event is both temporally and spatially regulated: the cell wall is degraded only after cell–cell contact and only in the region of cell–cell contact. To understand how cell fusion is regulated, we identified mutants defective in cell fusion based upon their defect in mating to a fus1 fus2 strain (Chenevert, J., N. Valtz, and I. Herskowitz. 1994. Genetics 136:1287–1297). Two of these cell fusion mutants are defective in the FPS1 gene, which codes for a glycerol facilitator (Luyten, K., J. Albertyn, W.F. Skibbe, B.A. Prior, J. Ramos, J.M. Thevelein, and S. Hohmann. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:1360–1371). To determine whether inability to maintain osmotic balance accounts for the defect in cell fusion in these mutants, we analyzed the behavior of an fps1Δ mutant with reduced intracellular glycerol levels because of a defect in the glycerol-3-phosphate dehydrogenase (GPD1) gene (Albertyn, J., S. Hohmann, J.M. Thevelein, and B.A. Prior. 1994. Mol. Cell. Biol. 14:4135– 4144): deletion of GPD1 partially suppressed the cell fusion defect of fps1 mutants. In contrast, overexpression of GPD1 exacerbated the defect. The fusion defect could also be partially suppressed by 1 M sorbitol. These observations indicate that the fusion defect of fps1 mutants results from inability to regulate osmotic balance and provide evidence that the osmotic state of the cell can regulate fusion. We have also observed that mutants expressing hyperactive protein kinase C exhibit a cell fusion defect similar to that of fps1 mutants. We propose that Pkc1p regulates cell fusion in response to osmotic disequilibrium. Unlike fps1 mutants, fus1 and fus2 mutants are not influenced by expression of GPD1 or by 1 M sorbitol. Their fusion defect is thus unlikely to result from altered osmotic balance.

Distribution of concanavalin a binding carbohydrates during mating in Chlamydomonas

1984 ◽  
Vol 66 (1) ◽  
pp. 223-239
Author(s):  
B.E. Millikin ◽  
R.L. Weiss
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Concanavalin A ◽  
Sodium Dodecyl ◽  
Fluorescein Isothiocyanate ◽  
Affinity Column ◽  
Wild Type ◽  
Mating Structure ◽  
Autolytic Activity ◽  
Surface Carbohydrates

Cell surface carbohydrates, detected by fluorescein isothiocyanate/concanavalin A (FITC-ConA), were identified at four locations on gametes of Chlamydomonas reinhardtii. (1) The cell wall: uniform labelling with FITC-ConA was observed; a substantial number of sites were localized in the sodium dodecyl sulphate-insoluble inner wall, which contains the flagellar collars. (2) The periplasm: a crescent-shaped area was visualized with FITC-ConA and localized by ferritin-ConA. We were able to recover autolytic activity on a ConA affinity column from the mating medium of wild-type cells after the release of these periplasmic sites. The cell-wall-less mutant CW15 displays no periplasmic sites and demonstrates a corresponding inability to release autolytic activity after mating for 60 min. A model for wall lysis is presented, which considers the involvement of these sites in the lytic process. (3) The mating structure: during mating a small fluorescent plaque-like site was observed on cells at a location corresponding to the carbohydrate-like zone of the mating type minus mating structure and may indicate the involvement of ConA binding material in gametic cell fusion. (4) Secreted products: following cell fusion zygotes begin to secrete ConA positive material at about 1 1/2 h. After 24 h a ConA positive zygote wall and pellicle appear.

scholarly journals Maintenance of Mating Cell Integrity Requires the Adhesin Fig2p

2002 ◽  
Vol 1 (5) ◽  
pp. 811-822 ◽  
Author(s):  
Mingliang Zhang ◽  
Daniel Bennett ◽  
Scott E. Erdman
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Large Family ◽  
Cell Integrity ◽  
Wild Type ◽  
Cell Agglutination ◽  
Spindle Positioning ◽  
Fungal Adhesins ◽  
Cell Cell

ABSTRACT Fungal adhesins represent a large family of serine/threonine-rich secreted glycoproteins. Adhesins have been shown to play roles in heterotypic and homotypic cell-cell adhesion processes, morphogenetic pathways and invasive/pseudohyphal growth, frequently in response to differentiation cues. Here we address the role of the Saccharomyces cerevisiae mating-specific adhesin Fig2p. Cells lacking FIG2 possess a variety of mating defects that relate to processes involving the cell wall, including morphogenetic defects, cell fusion defects, and alterations in agglutination activities. We found that mating-specific morphogenetic defects caused by the absence of FIG2 are suppressible by increased external osmolarity and that, during mating, fig2Δ cells display reduced viability relative to wild-type cells. These defects result from alterations in signaling activated by the mating and cell integrity pathways. Finally, we show that fig2Δ zygotes also have defects in zygotic spindle positioning that are osmoremedial, whereas the requirements for FIG2 in normal cell-cell agglutination and cell fusion during mating are insensitive to changes in the extracellular osmotic environment. We conclude that FIG2 performs distinct functions in the mating cell wall that are separable with respect to their ability to be suppressed by changes in external osmolarity and that a fundamental role of FIG2 in mating cells is the maintenance of cell integrity.

scholarly journals PAK Kinases Ste20 and Pak1 Govern Cell Polarity at Different Stages of Mating inCryptococcus neoformans

2004 ◽  
Vol 15 (10) ◽  
pp. 4476-4489 ◽  
Author(s):  
Connie B. Nichols ◽  
James A. Fraser ◽  
Joseph Heitman
Keyword(s):  
Sexual Identity ◽  
Cryptococcus Neoformans ◽  
Cell Polarity ◽  
Cell Fusion ◽  
Mating Type ◽  
Fungal Pathogen ◽  
Polar Growth ◽  
A Cells ◽  
P21 Activated Kinase ◽  
Cell Cell

Sexual identity and mating are linked to virulence of the fungal pathogen Cryptococcus neoformans. Cells of the α mating type are more prevalent and can be more virulent than a cells, and basidiospores are thought to be the infectious propagule. Mating in C. neoformans involves cell-cell fusion and the generation of dikaryotic hyphae, processes that involve substantial changes in cell polarity. Two p21-activated kinase (PAK) kinases, Pak1 and Ste20, are required for both mating and virulence in C. neoformans. We show here that Ste20 and Pak1 play crucial roles in polarized morphogenesis at different steps during mating: Pak1 functions during cell fusion, whereas Ste20 fulfills a distinct morphogenic role and is required to maintain polarity in the heterokaryotic mating filament. In conclusion, our studies demonstrate that PAK kinases are necessary for polar growth during mating and that polarity establishment is necessary for mating and may contribute to virulence of C. neoformans.

scholarly journals Generating heterokaryotic cells via bacterial cell-cell fusion

2021 ◽  
Author(s):  
Shraddha Shitut ◽  
Meng-Jie Shen ◽  
Bart Claushuis ◽  
Rico J. E. Derks ◽  
Martin Giera ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Tissue Repair ◽  
Genetic Recombination ◽  
Control Cell ◽  
Virus Transmission ◽  
Lipid Vesicles ◽  
Synthetic Cells ◽  
And Control ◽  
Cell Cell

Cell-cell fusion is fundamentally important for tissue repair, virus transmission, and genetic recombination, among other functions. Fusion has been mainly studied in eukaryotic cells and lipid vesicles, while cell-cell fusion in bacteria is less well characterized, due to the cell wall acting as a fusion-limiting barrier. Here we use cell wall-deficient bacteria to investigate the dynamics of cell fusion in bacteria that replicate without their cell wall. Stable, replicating cells containing differently labeled chromosomes were successfully obtained from fusion. We find that the rate of cell-cell fusion depends on the fluidity of cell membranes. Furthermore, we show that not only the efficiency but also the specificity of cell-cell fusion can be controlled via a pair of synthetic membrane-associated lipopeptides. Our results provide a molecular handle to understand and control cell-cell fusion to generate heterokaryotic cells, which was an important step in the evolution of protocells and of increasing importance for the design of synthetic cells.

scholarly journals Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.

1982 ◽  
Vol 92 (2) ◽  
pp. 378-386 ◽  
Author(s):  
U W Goodenough ◽  
P A Detmers ◽  
C Hwang
Keyword(s):  
Cell Fusion ◽  
Mating Type ◽  
Central Zone ◽  
Uranyl Acetate ◽  
Surface Coat ◽  
Wild Type ◽  
En Bloc ◽  
Mating Structure ◽  
Fusion Images ◽  
Coat Material

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.

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